Vancomycin kills Gram-positive bacteria by interfering with cell-wall biosynthesis. Recently, vancomycin-resistant enterococci and staphylococci have emerged. In the last grant period, we have proven that vancomycin analogs with activity against vancomycin-resistant strains bind to cell-wall bridges as well as the stem termini of cell-wall precursors. We have correlated bound structure and activity for these glycopeptide drugs and have characterized their modes of action using solid-state NMR detection of drug-complex formation and biosynthesis in whole cells of wild-type Staphylococcus aureus and Enterococcus faecium. In the next grant period, we plan to characterize the full 3D architecture of the peptidoglycan lattice of S. aureus and E. faecium, and the cell-wall binding sites of new glycopeptide and cyclic peptide drugs, using a combination of mass spectrometry (MS) and solid-state NMR. The MS experiments will provide insights into complex cell-wall structures with a much higher resolution than can be determined by NMR alone. The solid-state NMR will be used in two ways: first, to make quantitative determinations of average cell-wall composition that will guide and refine digestion and isolation procedures for the MS experiments; and second, to locate (by magic-angle spinning recoupling and spin-diffusion distance determinations) the bound drugs in the cell walls. New solid-state NMR analytical techniques will be developed in this effort, the most important of which is a T1 and T2 relaxation-compensated method for extracting distances from 13C-13C spin diffusion. These techniques will then be applied to the biomedically relevant characterization of new types of cyclic antimicrobial peptides, lipodecapeptides and lipo-octapeptides. The identification of their mode(s) of action and that of oritavancin (a vancomycin analogue which is near approval for clinical use) will be made in three types of vancomycin-resistant virulent strains of S. aureus and E. faecium: (i) vancomycin-resistant staph strains carrying the vanA gene which results in D-Ala-D-Lac peptidoglycan stem termini; (ii) vancomycin-resistant enterococcal strains also with the vanA gene but with the added complication of initially testing negative in all standard vancomycin-resistance assays used in the clinic; and (iii) intermediate-resistant staph strain Mu50 whose peptidoglycan-thickened cell wall resists treatment by both vancomycin and oritavancin but appears to be susceptible to at least one of the cyclic peptides.